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作者：Gal Y. Rocheforta, Bruno Delormeb, Adriana Lopezb, Olivier Hraultb, Pierre Bonneta, Pierre Charbordb, Vronique Edera, Jorge Domenechb / l# l3 q8 ^7 n; I
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; [* L3 x% v3 h; d1 l% b8 ~; ] 【摘要】: C/ K- W) J. I% P
MSCs constitute a population of multipotential cells giving rise to adipocytes, osteoblasts, chondrocytes, and vascular-smooth muscle-like hematopoietic supportive stromal cells. It remains unclear whether MSCs can be isolated from adult peripheral blood under stationary conditions and whether they can be mobilized in a way similar to hematopoietic stem cells. In this report, we show that MSCs are regularly observed in the circulating blood of rats and that the circulating MSC pool is consistently and dramatically increased (by almost 15-fold) when animals are exposed to chronic hypoxia. The immunophenotype and the adipocytic, osteoblastic, and chondrocytic differentiation potential of circulating MSCs were similar to those of bone marrow MSCs. Hypoxia-induced mobilization appears to be specific for MSCs since total circulating hematopoietic progenitor cells were not significantly increased. Our data provide an in vivo model amenable to analysis of MSC-mobilizing factors.
4 W" M6 _6 r! O3 o3 |# } 【关键词】 Stem cells Mobilization Blood Hypoxia Hematopoiesis" Q- b' f7 u4 \ R8 c, S
INTRODUCTION
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Mesenchymal stem cells (MSCs) constitute a population of multipotential cells giving rise to adipocytes, osteoblasts, chondrocytes, and vascular-smooth muscle-like hematopoietic supportive stromal cells .* I) B. V4 N$ `2 \( T
5 `, A; N$ g" U+ ^" EOne of the properties of stem cells is their capacity to home after infusion to the appropriate microenvironment(s) , which may be related to the low frequency of such cells at steady state. The cogent evidence that MSCs circulate into PB and can be mobilized into the bloodstream using appropriate agents such as cytokines would be of great therapeutic interest since it would allow the collection of MSCs from PB and the eventual manipulation of their subsequent homing to injured tissues.
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In this study, we found that MSCs are regularly observed in the circulating blood of rats and showed, to our knowledge for the first time, that the circulating pool is consistently and dramatically increased in animals subjected to chronic hypoxia. Our data provide an in vivo model amenable to analysis of MSC-mobilizing factors.
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) T) ~/ q+ {# F; ZMATERIALS AND METHODS
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Rat Model of Chronic Hypoxia
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Adult male Wistar rats (7 weeks, 220 g; Harlan, Gannat, France, http://www.harlan.com) were housed for 3 weeks in a hypoxic chamber (50 kPa), which caused chronic hypoxia, as previously described , and were compared with matched control rats housed in normoxic conditions (101 kPa). Three weeks later, PB and bone marrow (BM) samples were collected to evaluate cell counts and to characterize mesenchymal and hematopoietic progenitor cells.$ ?9 V5 f) V4 V2 z/ M8 g3 E
* ^/ N; L* y$ h9 _, {All animal investigations were carried out in accordance with the Guide for the Care and Use of Laboratory Animals published by the NIH and approved by the local ethics committee.
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Generation of MSCs
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5 z7 x0 B4 I* @9 XMSCs were obtained from rat femoral BM, as previously described ).- ?: ^# `7 i% S& d2 y" z
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Eight- to 12-ml peripheral blood samples were collected in heparinized tubes. Low-density mononuclear cells (MNCs) were separated on Ficoll-Hypaque density gradient (Amersham, Saclay Orsay, France, http://www.amersham.com), counted, and cultured as described for BM; cell density at culture inception was 106 cells per cm2; at P1, density was 10,000 cells per cm2.
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Mesenchymal Progenitor Cells. ^7 f5 s( `! T2 `2 q
& D; J1 z" _1 `/ w, ?! r" w* AFor colony-forming unit fibroblast (CFU-F) assays, BM total cells were plated per triplicate at densities of 5 x 104, 5 x 105, and 5 x 106 cells per 25-cm2 flask in proliferation culture medium, whereas for PB MNCs, cell densities were 105, 106, and 107 cells per 12.5-cm2 flask. The culture medium was changed on day 2, and adherent colonies (>50 cells) deriving from CFU-Fs were counted on days 6¨C10.
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$ H; _. q8 }! X5 G9 R2 o7 v2 |# J* ?Hematopoietic Progenitor Cells& J6 |- x5 l- f7 L
" w' |& m9 H: ^ l: d8 c$ CBM and PB MNCs were assayed for colony-forming units-granulocyte-macrophage (CFU-GM), colony-forming units-macrophage (CFU-M), colony-forming units-erythroid (CFU-E), burst-forming units-erythroid (BFU-E) and colony-forming units-mixed lineage (CFU-Mix) in semisolid culture medium. Briefly, MNCs were cultured in Iscove's modified Dulbecco's medium (Invitrogen) containing 1% (wt/vol) methylcellulose (Sigma-Aldrich, Saint Quentin Fallavier, France, http://www.sigmaaldrich.com), 1% (wt/vol) bovine serum albumin (Sigma-Aldrich), 1 x 10¨C4 M ß-mercaptoethanol (Sigma-Aldrich), and 30% FCS (Invitrogen). Cells were plated at 2.5 x 104 cells per milliliter for BM and 2.5 x 105 cells per milliliter for PB in 35-mm plastic dishes containing human G-CSF (100 IU/ml; AbCys, Paris, France, http://www.abcysonline.com), rat granulocyte-macrophage colony-stimulating factor (1,000 IU/ml; AbCys), rat stem cell factor (5 IU/ml; AbCys), rat interleukin-3 (1 IU/ml; AbCys), and rat erythropoietin (10 ng/ml; R&D Systems, Lille, France, http://www.rndsystems.com). Cells were incubated at 37¡ãC in a humidified 5% CO2 atmosphere, and colonies were scored on day 4 for CFU-E (aggregates >50 hemoglobinized cells), and on day 10 for CFU-GM, CFU-M (aggregates >50 nonhemoglobinized cells), BFU-E (aggregates >200 hemoglobinized cells), and CFU-Mix (aggregates >50 hemoglobinized and nonhemoglobinized cells).1 C6 f, l0 Q9 `6 Q
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MSC Immunophenotype
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2 ]0 Z! }# w6 n3 r4 M& h! g. E! NMembrane antigen expression on BM- and PB-derived MSCs was determined at P2 by flow cytometry with a FACSCalibur flow cytometer (Becton, Dickinson and Company, Le-Pont-de-Claix, France, http://www.bdbiosciences.com) using a 488-nm argon laser. Cells from single-cell suspension were incubated for 60 minutes at 4¡ãC with monoclonal antibodies (Abs) against rat antigens, including CD31 (clone TLD-3A12; Serotec, Cergy St. Christophe, France, http://www.serotec.com), CD44 (clone OX-50; Serotec), CD45 (clone MRC OX-1; Serotec), CD54 (clone 1A29; Serotec), CD73 (clone 5F/B9; Becton, Dickinson and Company) and CD90 (clone MRC OX-7; Serotec). Irrelevant isotype-identical Abs (clone F8¨C11-13; Serotec) served as negative control. Specific and unspecific Ab binding was detected with a secondary phycoerythrin-labeled anti-mouse Ab (Serotec). Samples were analyzed by collecting 10,000 events using Cell-Quest software (Becton, Dickinson and Company).
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MSC Differentiation# J$ D+ o+ ^' N" R' M2 H+ E
* L4 c* `9 U* ^: BP2 BM- and PB-derived MSC differentiation potential was evaluated as follows.
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9 W6 x H% C, }% ~. aAdipogenic Induction. Cells were cultured for 14 days in -MEM containing 10% (vol/vol) FCS, 100 µM isobutyl methylxanthine (Sigma-Aldrich), 60 µM indomethacin (Sigma-Aldrich), 1 µg/ml insulin (Sigma-Aldrich), and 0.5 µM hydrocortisone (Sigma-Aldrich), with medium changes every 3 days. Adipogenic differentiation was shown by cellular accumulation of large (5 µm in diameter) lipid vacuoles that were stained with oil red O (Sigma-Aldrich) and counterstained with 4,6-diamidino-2-phenylindole (AbCys).
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: G& b/ n" ], T* [+ i2 b9 p: wOsteogenic Induction. Cells were cultured for 21 days in -MEM containing 20% (vol/vol) FCS, 0.1 µM dexamethasone (Sigma-Aldrich), 2 mM ß-glycerophosphate (Sigma-Aldrich), and 150 µM ascorbic acid (Invitrogen); medium was changed every 3 days. Mineralization areas were revealed by von Kossa's stain.
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Chondrogenic Induction. Cells at 80% confluence were trypsinized with 0.05% (vol/vol) trypsin-EDTA and resuspended in low-glucose DMEM containing 1 mM dexamethasone (Sigma-Aldrich), 1 mM sodium pyruvate (Invitrogen), 1x insulin-transferrin-selenium (Invitrogen), 17 mM ascorbic acid (Invitrogen), 35 mM proline (Sigma-Aldrich), and 10 ng/ml transforming growth factor ß1 (R&D Systems). Viable cells were counted and seeded at a density of 5 x 105 cells per pellet in 15-ml conical tubes. Cells were gently centrifuged to the bottom of the tubes and allowed to form compact cell pellets, then incubated in a humidified atmosphere at 37¡ãC with 5% CO2 with medium changes every 3 days. After 21 days in culture, pellets were embedded in paraffin. Cartilage glycosaminoglycans were detected by staining with Safranin O (Sigma-Aldrich).' \. u5 K! h8 N5 ?# H. [
3 ?4 p* E. `6 F% }& O! [5 |Reverse Transcription-Polymerase Chain Reaction Analysis
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Total RNA was extracted from undifferentiated (control) and differentiated BM- and PB-derived MSCs using Trizol reagent (Invitrogen). A total of 100 ng of RNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) using SuperScript One-Step RT-PCR with Platinum Taq system (Invitrogen). Polymerase chain reaction was carried out with primers specific for rat acidic ribosomal phosphoprotein large P0 (Rplp0), lipoprotein lipase (Lpl), peroxisome proliferator-activated receptor 2 (Pparg2), bone -carboxyglutamate protein 2 (Bglap2), runt-related transcription factor 2 (Runx2), pro-1(II) collagen (Col2a1), and pro-1(X) collagen (Col10a1) (Table 1). Amplified cDNA fragments were electrophoresed through a 2% (wt/vol) agarose gel, stained by ethidium bromide, and photographed under an ultraviolet light transilluminator.
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Table 1. Primers used for reverse transcription-polymerase chain reactions/ w" b) ?" ]1 v, P A
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Statistical Analysis
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The values presented for each group are means ¡À SEM. Student's t test was used for comparison of mean values between different groups.
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6 v) t- D+ |: v: v" c5 c9 l. K& `RESULTS
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Hematological Parameters in Normoxic and Hypoxic Rats/ Z i. S; t5 J, F
; @6 W1 v% a0 J0 [Wistar rats were housed for 3 weeks in hypoxic conditions (n = 14), and none of them died; neither did control rats that were housed in normoxic conditions (n = 13). After 3 weeks, hematological parameters on PB samples were in close agreement with previous reports : hematocrit, hemoglobin values, and red blood cell counts were significantly increased (p & t$ L' k- _- o3 D' I4 k6 g7 i( a
0 Q ]9 \; f/ B$ O9 fTable 2. Hematological characteristics of peripheral blood samples from normoxic and hypoxic rats7 y( f6 s3 G) r4 C4 a
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Mesenchymal and Hematopoietic Progenitor Cells" q+ W6 u9 k$ w: Z: D
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Progenitor cell assays were performed on BM and PB samples from normoxic (BMN and PBN) and hypoxic (BMH and PBH) rats. As shown in Figure 1A, a dramatic increase in CFU-F frequency (of approximately 15-fold) was observed in the PB of rats subjected to chronic hypoxia (p , U) h3 }, u# x1 ^4 p6 _) H
3 ~4 T6 J9 p" t. _& iFigure 1. Mesenchymal and hematopoietic progenitor cell frequencies in bone marrow (BM) and peripheral blood (PB). (A): CFU-F frequency. Number of CFU-Fs (mean ¡À SEM) per 106 cells from seven BMN and PBN samples and eight BMH and PBH samples. * p / O) ]( P: h1 T, K
$ P+ |1 L! W: Y! H$ d/ B& B1 L; ]As shown in Figure 1B, the estimated total number of hematopoietic progenitor cells (HPCs) (mean ¡À SEM) per 106 MNCs was 8,860 ¡À 1,704 in BMN (n = 6), 11,501 ¡À 1,773 in BMH (n = 6), 35 ¡À 13 in PBN (n = 6) and 47 ¡À 18 in PBH (n = 6). These total numbers were not statistically different when comparing normoxic and hypoxic rats, although limited increase of circulating CFU-GM numbers were found after hypoxia (p
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Generation of MSCs Under Normoxic and Hypoxic Conditions
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. m+ [9 @4 g' `3 f- I, d2 sPB cells from normoxic and hypoxic rats, set in culture in parallel to BM cells from normoxic rats, formed, by the third week, a homogeneous layer of cells that closely resembled BM MSCs (Fig. 2A).% h. @. `$ Y" x4 d1 F
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Figure 2. Characteristics of BM and PB-derived cultured adherent cells. (A): Morphology. Passage 2 adherent cells from BMN, PBN, and PBH observed by phase contrast microscopy. Scale bar = 50 µM. (B): Immunophenotype. Fluorescence intensity histograms with specific antibodies (Abs) for membrane antigens (black line) and irrelevant isotypic-matched Ab as negative control (gray area). Experiments were performed in triplicate. Abbreviations: BMN, bone marrow of normoxic rats; MSC, mesenchymal stem cells; PBH, peripheral blood of hypoxic rats; PBN, peripheral blood of normoxic rats.
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: [5 ~/ G' O4 ]2 o1 B1 B' @The cell surface antigen expression of PBN- and PBH-derived adherent cells after two passages in culture was analyzed and compared with that of BM MSCs. The cultured adherent PBN- and PBH-derived cells were positive for CD44 (homing-associate cell adhesion molecule), CD54 (intercellular adhesion molecule-1), CD73 (ecto-5'-nucleotidase), and CD90 (Thy-1), but were negative for CD31 (platelet-endothelial cell adhesion molecule-1), CD45 (leukocyte common antigen) (Fig. 2B), and for CD18 (ß2 integrin), CD49d (4 integrin chain), and CD49f (6 integrin chain) (data not shown). The cell surface antigen expression pattern of PBN- and PBH-derived cells was therefore comparable to that of BM MSCs.
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To assess the capacity of PBN- and PBH-derived adherent cells to differentiate into mesenchymal lineages, we cultured the cells in adipogenic, osteogenic, and chondrogenic induction media for 14¨C21 days. We checked by specific stainings for the presence of cytoplasmic neutral lipid vacuoles, the formation of mineralized areas, and the accumulation of cartilage glycosaminoglycans. As shown in Figure 3A, PBN- and PBH-derived cells were positive for all specific markers, similarly to BM MSCs differentiated using identical protocols.8 s0 O: Y5 _* H3 K9 X6 J
* f" U* V2 o D$ iFigure 3. Mesenchymal stem cell differentiation potential of BM- and PB-derived adherent cells. (A): Histochemistry for Ad, Os, and Ch differentiation. Passage 2 BMN-derived (a, b, c), PBN-derived (d, e, f), and PBH-derived (g, h, i) adherent cells after culture in differentiation medium (Ad, Os, or Ch). Stains used were oil red O (a, d, g), Von Kossa's stain (b, e, h), and Safranin O stain (c, f, i). All experiments were performed in triplicate. Scale bar = 50 µm. (B): mRNA expression of lineage-specific genes. Passage 2 BMN-, PBN-, and PBH-derived adherent cells after culture in differentiation medium (Ad, Os, or Ch) or in Ctl proliferation medium. All experiments were performed in triplicate. Abbreviations: Ad, adipogenic; Bglap2, bone -carboxyglutamate protein 2; BMN, bone marrow of normoxic rats; Ch, chondrogenic; Col2a1, pro1(II) collagen; Coll0a1, pro-1(X) collagen; Ctl, control; Lpl, lipoprotein lipase; Neg, negative (polymerase chain reaction without cDNA); Os, osteogenic; PBH, peripheral blood of hypoxic rats; PBN, peripheral blood of normoxic rats; Pparg2, peroxisome proliferator-activated receptor 2; Rplp0, ribosomal phosphoprotein large P0 (Rplp0).! z: U0 u. H# [0 o5 @0 ]
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In addition, PBN- and PBH-derived cells expressed mRNAs of Lpl and Pparg2 (adipocytic markers), Bglap and Runx2 (osteoblastic markers), and Col2a1 and Col10a1 (chondrocytic markers), as did BM MSCs (Fig. 3B).
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/ e: R4 B; j/ T( y/ l, LThese results show that PB-derived adherent cells collected from normoxic and hypoxic rats display a trilineage-differentiation potential comparable to that of BM MSCs. Taken together, our results (CFU-Fs, differentiation potential, and immunophenotype) indicate that blood-derived adherent cells are bona fide MSCs.
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) N# [( Y+ \5 L2 p* }DISCUSSION
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This study indicates that a small number of MSCs consistently circulate in the PB under stationary conditions and that the circulating pool is greatly increased by hypoxia. Remarkably, this increase is relatively specific for MSCs, since HPCs showed no or little increase under hypoxic conditions.
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Several studies have been carried out to isolate MSCs in humans and in animals from adult PB using culture conditions equivalent to those used for BM-derived MSCs. Some studies concluded that cells similar to BM MSCs are present in PB from humans and animals . In the present study, we report that low levels of multipotent MSCs were detected in the PB of adult rats at steady state. Moreover, we show, to our knowledge for the first time, that the circulating blood MSC pool is dramatically increased in hypoxic animals.
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" I: \; h& `3 Y/ b) |3 EMechanisms of MSC mobilization into the bloodstream are unknown. They are probably different from those involved in growth factor-induced HSC mobilization, although some reports indicate that MSCs can be detected directly or indirectly in PB grafts after such mobilization procedure , CFU-Fs are not detected in the blood of large series of patients after G-CSF infusion. The mobilizing effect of hypoxia observed in our study concerned selective by CFU-Fs, since total circulating HPC level was not significantly increased. The BM CFU-F frequency was unchanged, suggesting that hypoxia either favors MSC egress from the BM into the bloodstream without significantly increasing the BM pool or induces the mobilization of MSCs from other non-BM sources.
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4 A4 b- G3 I9 `& u l. Y( ?Recent studies have demonstrated that long-term cultures of MSCs under an atmosphere of low oxygen that closely approximates documented in vivo oxygen tension enabled these cells to optimally proliferate, to differentiate . Our animal model provides the opportunity to test in vivo the role of such factors in the mobilization process of MSCs. Determination of the critical factors responsible for this process is of clinical importance since they could be used further to trigger at will the mobilization of endogenous MSCs without the cumbersome cell culture expansion step, a potential source of contamination or transformation.* T% A9 e/ Q% x* M$ p
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In conclusion, we report that few MSCs circulate in the PB under stationary conditions in rats. Large amounts of MSCs can be mobilized (an increase of almost 15-fold) under hypoxic conditions. These data demonstrate that MSCs can be mobilized into the PB via stimuli distinct from those involved in hematopoietic stem cell mobilization.# Q# b9 N" {! U
: M& V) L4 ~8 b, n5 I: c, e! FDISCLOSURES" C2 v! E0 T- t, U% l) ]' Z8 _
2 \; r: e% K6 o" O+ {/ g, ~- eThe authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS
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7 `. {6 Y9 A3 I2 l- b* ~" u# vWe are grateful to Elfi Ducrocq and Marie-Christine Bernard for expert technical assistance. This work was supported by grants from the French Regional Council of Centre and the European integrated project FIRST (Further Improvement of Radiotherapy of cancer through Side effect reduction by application of stem cell Transplantation Contract 503436).) S# g, I- r2 P
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